Methods of treating exercise-induced pulmonary hemorrhage

ABSTRACT

Provided herein are methods of treating EIPH in a subject in need thereof. The methods comprise administering an effective amount of a pharmaceutical composition to the subject. In some embodiments, the pharmaceutical composition comprises a granuloma fluid, or a composition derived from a granuloma fluid. In some embodiments, the pharmaceutical composition comprises a cell. These pharmaceutical compositions may be administered by any suitable route of administration, and may be administered with one or more further therapeutic agents. Also provided are kits comprising pharmaceutical compositions for use with the methods provided herein.

CROSS REFERENCE

The present application is a continuation of U.S. patent applicationSer. No. 15/329,220 filed Jan. 25, 2017, which is the U.S. nationalstage of International Application no. PCT/US2015/042105, filed Jul. 24,2015, which claims the benefit of U.S. provisional application No.62/029,310, filed Jul. 25, 2014, the content of each is content of whichis hereby incorporated by reference in its entirety.

FIELD

Provided herein are methods for treating exercise-induced pulmonaryhemorrhage in a subject. In some embodiments, the methods comprise theadministration of a granuloma fluid to a subject in need thereof. Insome embodiments, the methods comprise administration of a cell to asubject in need thereof.

BACKGROUND

Exercise-induced pulmonary hemorrhage (EIPH), also known as “bleeding”or a “bleeding attack,” refers to the accumulation of blood in theairways of the lung in association with exercise. EIPH is common inhorses undertaking intense exercise, but it has also been reported inhuman athletes, racing camels, and racing greyhounds. In thoroughbredracehorses, the estimated prevalence of EIPH is 43 to 75%. See Pascoe etal., Amer. J. Vet. Res., 1981, 42:703-707, incorporated by reference inits entirety.

Animals with EIPH may be referred to as “bleeders” or as having “brokena blood vessels.” In the majority of cases EIPH is not apparent unlessan endoscopic examination of the airways is performed followingexercise. However, some subjects may show bleeding at the nostrils afterexercise, known as epistaxis.

A number of treatments have been used or suggested for EIPH, includingresting, anti-inflammatorics (e.g., corticostcroids), bronchodilators,anti-hypertensive agents (including nitric oxide donors andphosphodiesterase inhibitors), conjugated estrogens (e.g., Premarin®),antifibrinolytics (e.g., aminocaproic acid and tranexamic acid), snakevenom, aspirin, vitamin K, bioflavinoids, diuretics (e.g., furosemide,known as Lasix® or Salix), nasal strips, and omega-3 fatty acids.

Although furosemide is the most common treatment used in race horses, itis believed to be ineffective in a large number of subjects. Furosemidemay also improve racing times in horses both with and without EIPH,possibly due to a lowering of body weight as a consequence of its potentdiuretic action. The use of furosemide in competing horses is thereforeprohibited in some countries, and it is regarded as a banned substanceby the International Olympic Committee. Moreover, chronic usage of thedrug can lead to hypokalemia and hypomagnesemia. Finally, the diureticeffects of furosemide can lead to dehydration, which can be detrimentalto the health of subjects engaging in athletic activities.

There is therefore a need for new treatments for EIPH, with improvedefficacy and fewer side-effects.

SUMMARY

Provided herein are methods of treating EIPH in a subject in needthereof.

In some embodiments, the method comprises administering an effectiveamount of a pharmaceutical composition comprising granuloma fluid, or acomposition derived therefrom, to the subject. In some embodiments, themethod comprises administering an effective amount of a pharmaceuticalcomposition comprising a cell to the subject. In some embodiments, thecell is a stem cell. In some embodiments, the stem cell is a mesenchymalstem cell.

The pharmaceutical compositions can be administered systemically orlocally. In some embodiments, the pharmaceutical composition isadministered by a route of administration selected from the nasal,pulmonary, inhalation, intraarterial, intradermal, intramuscular,intraperitoneal, intravenous, parenteral, and subcutaneous routes. Insome embodiments, the pharmaceutical compositions arc administered by aroute of administration selected from the nasal and pulmonary routes.

In some embodiments, the subject is selected from a horse, a dog, acamel, a monkey, a cat, a pig, a cow, a goat, a llama, a sheep, a mouse,a rat, a rabbit, and a human.

In some embodiments, the pharmaceutical composition is administered incombination with at least one further active agent.

Also provided are kits for carrying out the methods of treatmentprovided herein. In some embodiments, the kit comprises an effectiveamount of a pharmaceutical composition comprising granuloma fluid, or acomposition derived therefrom. In some embodiments, the kit comprises aneffective amount of a cell. In some embodiments, the cell is a stemcell. In some embodiments, the stem cell is a mesenchymal stem cell. Insome embodiments, the kit comprises a nasal strip.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein are methods of treating EIPH in a subject in needthereof. The methods comprise administering an effective amount of apharmaceutical composition to the subject. In some embodiments, thepharmaceutical composition comprises a granuloma fluid, or a compositionderived from a granuloma fluid. In some embodiments, the pharmaceuticalcomposition comprises a cell. In some embodiments, the pharmaceuticalcomposition comprises a granuloma fluid, or a composition derived from agranuloma fluid, and a cell. As described further elsewhere in thisdisclosure, these pharmaceutical compositions may be administered by anysuitable route of administration, and may be administered with one ormore further therapeutic agents. Also provided are kits comprisingpharmaceutical compositions for use with the methods provided herein.

1. Definitions

As used herein, the term “about” refers to the stated value plus orminus 15%. For example, a value of “about 10” encompasses a range of 8.5to 11.5.

As used herein, the term “granuloma fluid” refers to a fluid that iscollected from the site of a granuloma. A granuloma forms when theimmune system attempts to isolate a substance that it perceives asforeign but that it is unable to eliminate. A granuloma may be induced,for example, by implantation of a foreign body in a mammal. Fluid fromthe granuloma (“granuloma fluid”) can be collected, for example, byutilizing a fluid collection device as the foreign body. The collectiondevice can be introduced into any suitable tissue of an animal, such asan omentum or a subcutaneous tissue. The process of inducing granulomaformation and collecting fluid therefrom is described in more detailelsewhere in this disclosure and, for example, in U.S. Pat. Pub. No.2011/0038903 A1, incorporated by reference in its entirety. Thegranuloma fluid resembles plasma, with respect to the major proteins(e.g., albumin and globulins), when analyzed by sodium dodecylsulfate—polyacrylamide gel electrophoresis (SDS-PAGE).

As used herein, the term “composition derived from granuloma fluid”includes, for example, a powder produced from the granuloma fluid, aconcentrated granuloma fluid, or a diluted granuloma fluid. A powder maybe produced from granuloma fluid by, for example, drying (e.g.,lyophilization), or by any other suitable method known in the art. Aconcentrated granuloma fluid may be produced by, for example,centrifugal filtration, or by any other suitable method known in theart. A diluted granuloma fluid may be produced by, for example, additionof a solvent to a granuloma fluid, or by any other suitable method knownin the art.

As used herein, the term “granuloma powder” refers to a powder producedby drying the granuloma fluid. Generally, each milliliter of granulomafluid yields about 5 to about 200 mg of granuloma powder. In someaspects, each milliliter of granuloma fluid yields about 10 to about 100mg of granuloma powder. In some aspects, each milliliter of granulomafluid yields about 25 to about 75 mg of granuloma powder. In someaspects, each milliliter of granuloma fluid yields about 75 to about 100mg of granuloma powder. In some embodiments, each milliliter ofgranuloma fluid yields about 50 mg of granuloma powder. In some aspects,each milliliter of granuloma fluid yields about 75 mg of granulomapowder.

As used herein, the term “granuloma cells” refers to the cells that arepresent at the site of a granuloma. These cells include any cell typethat is present at the site of the granuloma, including stem cells(e.g., omental stem cells, subcutaneous granulation tissue stem cells,bone marrow derived mesenchymal stem cells), progenitor cells, or anyother cell found at the site of a granuloma.

As used herein, “treating” or “treatment” of a disease or disorderrefers, in certain embodiments, to ameliorating a disease or disorderthat exists in a subject. In some embodiments, “treating” or “treatment”includes ameliorating at least one physical parameter, which may beindiscernible by the subject. In some embodiments, “treating” or“treatment” includes modulating the disease or disorder, eitherphysically (e.g., stabilization of a discernible symptom) orphysiologically (e.g., stabilization of a physical parameter) or both.In some embodiments, “treating” or “treatment” includes delaying orpreventing the onset of the disease or disorder. In some embodiments,“treating” or “treatment” includes mitigating the incidence of a diseaseor disorder, by periodic administration of a pharmaceutical composition.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of a composition provided hereinthat is useful for treating or preventing EIPH, or a symptom thereof. Insome embodiments, the symptom is selected from epistaxis, blood in thepulmonary system, pulmonary hypertension, decrease in athleticperformance, abnormal breathing during exercise, repeated swallowing,lack of appetite and combinations thereof.

As used herein, the term “subject’ means a mammalian subject. Exemplarysubjects include, but are not limited to horses, monkeys, dogs, cats,mice, rats, rabbits, camels, cows, goats, llamas, sheep, and humans. Incertain embodiments, the subject is a horse. In some embodiments, thesubject is a human. In some embodiments, the subject has been diagnosedwith EIPH. In some embodiments, the subject is suspected to have EIPH.In some embodiments, the subject is at risk for developing EIPH. In someembodiments, a subject with EIPH is identified by epistaxis. In someembodiments, the subject is identified by endoscopy, bronchoalvcolarlavagc, biopsy, radiograph, and/or pulmonary scintigraphy. In someembodiments, a subject at risk for developing EIPH is identified by ahistory of EIPH, or by an elevated pulmonary blood pressure.

2. Methods of Treating MPH

Provided herein are methods of treating EIPH in a subject in needthereof. The methods comprise administering an effective amount of apharmaceutical composition to the subject. In some embodiments, thepharmaceutical composition comprises a granuloma fluid, or a compositionderived from a granuloma fluid. In some embodiments, the pharmaceuticalcomposition comprises a cell. In some embodiments, the cell is a stemcell. In some embodiments, the stem cell is a mesenchymal stem cell.

In some embodiments, the pharmaceutical composition comprises agranuloma fluid, or a composition derived from a granuloma fluid.Accordingly, in some embodiments, provided herein are methods oftreating EIPH in a subject in need thereof by administering an effectiveamount of a pharmaceutical composition comprising a granuloma fluid, ora composition derived from a granuloma fluid, to the subject. Thepharmaceutical composition comprising the granuloma fluid, orcomposition derived therefrom, can be administered in any suitable doseor by any suitable route of administration. It can also be administeredwith any suitable further agent. Suitable doses, routes ofadministration, and further agents are described elsewhere in thisdisclosure.

In some embodiments, the pharmaceutical composition comprises a cell.Accordingly, in some embodiments, provided herein arc methods oftreating EIPH in a subject in need thereof by administering an effectiveamount of a pharmaceutical composition comprising a cell to the subject.The pharmaceutical composition comprising the cell can be administeredin any suitable dose or by any suitable route of administration. It canalso be administered with any suitable further agent. Suitable doses,routes of administration, and further agents are described elsewhere inthis disclosure.

In some embodiments, the pharmaceutical composition comprises a stemcell. Accordingly, in some embodiments, provided herein are methods oftreating EIPH in a subject in need thereof by administering an effectiveamount of a pharmaceutical composition comprising a stem cell to thesubject. The pharmaceutical composition comprising the stem cell can beadministered in any suitable dose or by any suitable route ofadministration. It can also be administered with any suitable furtheragent. Suitable doses, routes of administration, and further agents aredescribed elsewhere in this disclosure.

In some embodiments, the pharmaceutical composition comprises amesenchymal stem cell. Accordingly, in some embodiments, provided hereinarc methods of treating EIPH in a subject in need thereof byadministering an effective amount of a pharmaceutical compositioncomprising a mesenchymal stem cell to the subject. The pharmaceuticalcomposition comprising the mesenchymal stem cell can be administered inany suitable dose or by any suitable route of administration. It canalso be administered with any suitable further agent. Suitable doses,routes of administration, and further agents arc described elsewhere inthis disclosure.

In some embodiments, the pharmaceutical composition comprises agranuloma fluid, or a composition derived from a granuloma fluid and acell. Accordingly, in some embodiments, provided herein are methods oftreating EIPH in a subject in need thereof by administering an effectiveamount of a pharmaceutical composition comprising a granuloma fluid, ora composition derived from a granuloma fluid, and a cell to the subject.The pharmaceutical composition comprising the granuloma fluid, orcomposition derived from granuloma fluid, and cell can be administeredin any suitable dose or by any suitable route of administration. It canalso be administered with any suitable further agent. Suitable doses,routes of administration, and further agents are described elsewhere inthis disclosure. In some embodiments, the cell is a stem cell. In someembodiments, the stem cell is a mesenchymal stem cell.

In some embodiments, provided are methods of treating EIPH byadministering an amount of a pharmaceutical composition that iseffective to reduce the severity of EIPH on a grading system. Anysuitable grading system may be used to evaluate EIPH. In someembodiments, the grading system is an endoscopic grading system thatranges from 0 to 4, with the following characteristics: Grade 0: noblood detected; Grade 1: one or more flecks of blood or <2 short, narrowstreams (<10% of tracheal surface); Grade 2: one long stream or >2 shortstreams occupying less than 33% of tracheal circumference; Grade 3:multiple distinct streams of blood, covering greater than 33% oftracheal circumference; and Grade 4: multiple, coalescing streams ofblood, covering more than 90% of the tracheal surface with blood poolingat the thoracic inlet. See Pascoe et al., Amer. I Vet. Res., 1981,42:703-707; Mason et al., “Exercise-induced pulmonary haemorrhage inhorses,” in D. H. Snow et al. (Eds.), Equine Exercise Physiology, pp.57-63, Granata Editions, Cambridge; Pascoe et al., Amer. I Vet. Res.,1985, 46:2000-2003; and Lapointe et al., Equine Vet. J., 1994,26:482-485, each of which is incorporated by reference in its entirety.

In some aspects, the severity of EIPH is reduced from a 4 to a 0. Insome aspects, the severity of EIPH is reduced from a 4 to a 1. In someaspects, the severity of EIPH is reduced from a 4 to a 2. In someaspects, the severity of EIPH is reduced from a 4 to a 3.

In some aspects, the severity of E1PH is reduced from a 3 to a 0. Insome aspects, the severity of EIPH is reduced from a 3 to a 1. In someaspects, the severity of EIPH is reduced from a 3 to a 2.

In some aspects, the severity of EIPH is reduced from a 2 to a 0. Insome aspects, the severity of EIPH is reduced from a 2 to a 1.

In some aspects, the severity of EIPH is reduced from a 1 to a 0.

3. Doses, Unit Dosage Forms, and Dosing Schedules

The clinician (e.g., veterinarian or physician) will determine the dose,unit dosage form, and dosing schedule that is most appropriate for thesubject based on, for example, whether the treatment is preventative orcurative, and the age, weight, and disease state of the subject.Illustrative doses, unit dosage forms, and dosing schedules are providedbelow.

3.1. Doses of Granuloma Fluid and Compositions Derived Therefrom, andUnit Dosage Forms Comprising Granuloma Fluid and Compositions DerivedTherefrom

As discussed above, a particular volume of granuloma fluid generallyyields a particular mass of granuloma powder when dried. The mass ofgranuloma powder remaining when the granuloma fluid is dried varies, butis generally in the range of about 5 mg to about 200 mg per milliliterof granuloma fluid, more particularly from about 25 mg to about 75 mgper milliliter of granuloma fluid. Accordingly, in some embodiments, thedose of granuloma fluid, or composition derived therefrom, can becalculated based on an equivalent dose of granuloma powder.

For example, if a pharmaceutical composition comprises granuloma fluiditself, then a 1 milliliter dose of granuloma fluid may comprise anequivalent does of granuloma powder that ranges from about 5 mg to about200 mg, or more particularly from about 25 mg to about 75 mg. On theother hand, if the pharmaceutical composition comprises granulomapowder, then the dose can be determined based on the mass of thegranuloma powder itself. The equivalent dose of granuloma powder for agranuloma fluid can be determined by drying the granuloma fluid andweighing the resulting powder.

In some embodiments, the pharmaceutical composition comprises granulomafluid. In some embodiments, the pharmaceutical composition comprisesgranuloma powder. In some embodiments, the pharmaceutical compositioncomprises a concentrated granuloma fluid. In some embodiments, thepharmaceutical composition comprises a diluted granuloma fluid.

In some embodiments, the dose of granuloma fluid, or composition derivedtherefrom, administered to the subject is equivalent to about 1 mg toabout 20 g of granuloma powder. In some aspects, the dose is equivalentto about 10 mg to about 20 g of granuloma powder. In some aspects, thedose is equivalent to about 100 mg to about 20 g of granuloma powder. Insome aspects, the dose is equivalent to about 500 mg to about 20 g ofgranuloma powder. In some aspects, the dose is equivalent to about 1 gto about 20 g of granuloma powder. In some aspects, the dose isequivalent to about 5 g to about 20 g of granuloma powder. In someaspects, the dose is equivalent to about 10 g to about 20 g of granulomapowder. In some aspects, the dose is equivalent to about 1 mg to about10 g of granuloma powder. In some aspects, the dose is equivalent toabout 1 mg to about 5 g of granuloma powder. In some aspects, the doseis equivalent to about 1 mg to about 1 g of granuloma powder. In someaspects, the dose is equivalent to about 1 mg to about 500 mg ofgranuloma powder. In some aspects, the dose is equivalent to about 1 mgto about 100 mg of granuloma powder. In some aspects, the dose isequivalent to about 1 mg to about 10 mg of granuloma powder. In someaspects, the dose is equivalent to about 200 mg to about 5 g ofgranuloma powder. In some aspects, the dose is equivalent to about 200mg to about 2.5 g of granuloma powder. In some aspects, the dose isequivalent to about 200 mg to about 1 g of granuloma powder.

In some embodiments, the dose of granuloma fluid, or composition derivedtherefrom, may vary depending on the body weight of the subject. In someembodiments, the dose is equivalent to about 0.01 mg/kg to about 1 g/kgof granuloma powder. In some aspects, the dose is equivalent to about0.1 mg/kg to about 1 g/kg of granuloma powder. In some aspects, the doseis equivalent to about 1 mg/kg to about 1 g/kg of granuloma powder. Insome aspects, the dose is equivalent to about 5 mg/kg to about 1 g/kg ofgranuloma powder. In some aspects, the dose is equivalent to about 10mg/kg to about 1 g/kg of granuloma powder. In some aspects, the dose isequivalent to about 50 mg/kg to about 1 g/kg of granuloma powder. Insome aspects, the dose is equivalent to about 100 mg/kg to about 1 g/kgof granuloma powder. In some aspects, the does is equivalent to about250 mg/kg to about 1 g/kg of granuloma powder. In some aspects, the doesis equivalent to about 500 mg/kg to about 1 g/kg of granuloma powder. Insome aspects, the dose is equivalent to about 0.1 mg/kg to about 500mg/kg of granuloma powder. In some aspects, the dose is equivalent toabout 0.1 mg/kg to about 250 mg/kg of granuloma powder. In some aspects,the dose is equivalent to about 0.1 mg/kg to about 100 mg/kg ofgranuloma powder. In some aspects, the dose is equivalent to about 0.1mg/kg to about 50 mg/kg of granuloma powder. In some aspects, the doseis equivalent to about 0.1 mg/kg to about 25 mg/kg of granuloma powder.In some aspects, the dose is equivalent to about 0.1 mg/kg to about 10mg/kg of granuloma powder. In some aspects, the dose is equivalent toabout 0.1 mg/kg to about 5 mg/kg of granuloma powder. In some aspects,the dose is equivalent to about 0.1 mg/kg to about 1 mg/kg of granulomapowder.

In some embodiments, the pharmaceutical composition is in the form of aunit dosage that comprises a particular dose of granuloma fluid, orcomposition derived therefrom. The amount of granuloma fluid, orcomposition derived therefrom, in the unit dosage form will varydepending on the route of administration and other factors recognized byone of skill in the art.

In some embodiments, the unit dosage form comprises a dose equivalent toabout 10 mg to about 20 g of granuloma powder. In some aspects, the unitdosage form comprises a dose equivalent to about 50 mg to about 20 g ofgranuloma powder. In some aspects, the unit dosage form comprises a doseequivalent to about 100 mg to about 20 g of granuloma powder. In someaspects, the unit dosage form comprises a dose equivalent to about 250mg to about 20 g of granuloma powder. In some aspects, the unit dosageform comprises a dose equivalent to about 500 mg to about 20 g ofgranuloma powder. In some aspects, the unit dosage form comprises a doseequivalent to about 1 g to about 20 g of granuloma powder. In someaspects, the unit dosage form comprises a dose equivalent to about 5 gto about 20 g of granuloma powder. In some aspects, the unit dosage formcomprises a dose equivalent to about 10 g to about 20 g of granulomapowder. In some aspects, the unit dosage form comprises a doseequivalent to about 15 g to about 20 g of granuloma powder. In someaspects, the unit dosage form comprises a dose equivalent to about 50 mgto about 15 g of granuloma powder. In some aspects, the unit dosage formcomprises a dose equivalent to about 50 mg to about 10 g of granulomapowder. In some aspects, the unit dosage form comprises a doseequivalent to about 50 mg to about 5 g of granuloma powder. In someaspects, the unit dosage form comprises a dose equivalent to about 50 mgto about 1 g of granuloma powder. In some aspects, the unit dosage formcomprises a dose equivalent to about 50 mg to about 500 mg of granulomapowder. In some aspects, the unit dos-age form comprises a doseequivalent to about 50 mg to about 250 mg of granuloma powder. In someaspects, the unit dosage form comprises a dose equivalent to about 50 mgto about 100 mg of granuloma powder. In some aspects, the unit dosageform comprises a dose equivalent to about 5 mg, 10 mg, 25 mg, 50 mg, 100mg, 250 mg, 500 mg, 1 g, 5 g, 10 g, 15 g, or 20 g of granuloma powder.

3.2. Doses of Cells and Unit Dosage Forms Comprising Cells

In some embodiments, the pharmaceutical composition comprises cells. Insome embodiments, the Cells arc stem cells. In some embodiments, thestem cells arc mescnchymal stem cells.

In some embodiments, the dose of cells administered to the subject isabout 5×10⁴ to about 5×10¹¹ cells. In some aspects, the dose of cellsadministered to the subject is about 5×10⁴ to about 5×10¹⁰ cells. Insome aspects, the dose of cells administered to the subject is about5×10⁴ to about 5×10⁹ cells. In some aspects, the dose of cellsadministered to the subject is about 5×10⁴ to about 5×10⁸ cells. In someaspects, the dose of cells administered to the subject is about 5×10⁴ toabout 5×10⁷ cells. In some aspects, the dose of cells administered tothe subject is about 5×10⁴ to about 5×10⁶ cells. In some aspects, thedose of cells administered to the subject is about 5×10⁴ to about 5×10⁵cells. In some aspects, the dose of cells administered to the subject isabout 5×10⁵ to about 5×10¹¹ cells. In some aspects, the dose of cellsadministered to the subject is about 5×10⁶ to about 5×10¹¹ cells. Insome aspects, the dose of cells administered to the subject is about5×10⁷ to about 5×10¹¹ cells. In some aspects, the dose of cellsadministered to the subject is about 5×10⁸ to about 5×10¹¹ cells. Insome aspects, the dose of cells administered to the subject is about5×10⁹ to about 5×10¹¹ cells. In some aspects, the dose of cellsadministered to the subject is about 5×10¹⁰ to about 5×10¹¹ cells.

In some embodiments, the dose of cells may vary depending on the bodyweight of the subject. In some embodiments, the dose is equivalent toabout 5×10⁴ cells/kg to about 5×10¹¹ cells/kg. In some aspects, the doseis equivalent to about 5×10⁵ cells/kg to about 5×10¹¹ cells/kg. In someaspects, the dose is equivalent to about 5×10⁶ cells/kg to about 5×10¹¹cells/kg. In some aspects, the dose is equivalent to about 5×10⁷cells/kg to about 5×10¹¹ cells/kg. In some aspects, the dose isequivalent to about 5×10⁸ cells/kg to about 5×10¹¹ cells/kg. In someaspects, the dose is equivalent to about 5×10⁹ cells/kg to about 5×10¹¹cells/kg. In some aspects, the dose is equivalent to about 5×10¹⁰cells/kg to about 5×10¹¹ cells/kg. In some aspects, the dose isequivalent to about 5×10⁴ cells/kg to about 5×10¹⁰ cells/kg. In someaspects, the dose is equivalent to about 5×10⁴ cells/kg to about 5×10⁹cells/kg. In some aspects, the dose is equivalent to about 5×10⁴cells/kg to about 5×10⁸ cells/kg. In some aspects, the dose isequivalent to about 5×10⁴ cells/kg to about 5×10⁷ cells/kg. In someaspects, the dose is equivalent to about 5×10⁴ cells/kg to about 5×10⁶cells/kg. In some aspects, the dose is equivalent to about 5×10⁴cells/kg to about 5×10⁵ cells/kg.

In some embodiments, the pharmaceutical composition is in the form of aunit dosage that comprises a particular dose of cells. The number ofcells in the unit dosage form will vary depending on the route ofadministration and other factors recognized by one of skill in the art.

In some embodiments, the unit dosage form comprises about 5×10⁴ to about5×10¹¹ cells. In some aspects, the unit dosage form comprises about5×10⁵ to about 5×10¹¹ cells. In some aspects, the unit dosage formcomprises about 5×10⁶ to about 5×10¹¹ cells. In some aspects, the unitdosage form comprises about 5×10⁷ to about 5×10¹¹ cells. In someaspects, the unit dosage form comprises about 5×10⁸ to about 5×10¹¹cells. In some aspects, the unit dosage form comprises about 5×10⁹ toabout 5×10¹¹ cells. In some aspects, the unit dosage form comprisesabout 5×10¹⁰ to about 5×10¹¹ cells. In some aspects, the unit dosageform comprises about 5×10⁴ to about 5×10¹⁰ cells. In some aspects, theunit dosage form comprises about 5×10⁴ to about 5×10⁹ cells. In someaspects, the unit dosage font' comprises about 5×10⁴ to about 5×10⁸cells. In some aspects, the unit dosage form comprises about 5×10⁴ toabout 5×10⁷ cells. In some aspects, the unit dosage form comprises about5×10⁴ to about 5×10⁶ cells. In some aspects, the unit dosage formcomprises about 5×10⁴ to about 5×10⁵ cells.

3.3. Dosing Schedules

The pharmaceutical composition may be administered to a subjectaccording to any suitable dosing schedule. In some embodiments, thepharmaceutical composition is administered once. In some embodiments,the pharmaceutical composition is administered more than once.

In some embodiments, the pharmaceutical composition is administeredaccording to a particular frequency. In some embodiments, the frequencyis daily, every 2 days, every 3 days, every 4 days, every 5 days, every6 days, every 7 days, every 8 days, every 9 days, every 10 days, every11 days, every 12 days, every 13 days, or every 14 days. In someembodiments, the frequency is every 3 weeks, every 4 weeks, every 5weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every10 weeks, every 11 weeks, or every 12 weeks. In some embodiments, thefrequency is every 1 month, every 2 months, every 3 months, every 4months, every 5 months, every 6 months, every 7 months, every 8 months,every 9 months, every 10 months, every 11 months, every 12 months, every13 months, every 14 months, every 15 months, every 16 months, every 17months, or every 18 months. In some embodiments, the frequency is every2 years, every 3 years, every 4 years, or every 5 years.

In some embodiments, the subject is treated over a particular durationof time. In some embodiments, the duration of treatment is from about 1week to about 10 years. In some aspects, the duration of treatment isfrom about 1 week to about 5 years. In some aspects, the duration oftreatment is from about 1 week to about 1 year. In some aspects, theduration of treatment is from about 1 week to about 6 months. In someaspects, the duration of treatment is from about 1 week to about 3months. In some aspects, the duration of treatment is from about 1 weekto about 1 month. In some aspects, the duration of treatment is fromabout 3 months to about 5 years. In some aspects, the duration oftreatment is from about 6 months to about 5 years. In some aspects, theduration of treatment is from about 1 year to about 5 years.

Each dose may be administered over any suitable period of time. In someembodiments, the dose is administered as a bolus dose. In someembodiments, the dose is administered over a period of about 1 minute toabout 4 hours. In some aspects, the dose is administered over a periodof about 1 minute to about 2 hours. In some aspects, the dose isadministered over a period of about 1 minute to about 1 hour. In someaspects, the dose is administered over a period of about 1 minute toabout 30 minutes. In some aspects, the dose is administered over aperiod of about 1 minute to about 15 minutes.

In some embodiments, a dose is administered to a horse after a race. Insome embodiments, a dose is administered to a horse periodically betweenraces.

4. Routes of Administration

The methods provided herein may be carried out by administering thepharmaceutical compositions by any suitable routes of administration.The route of administration can be local or systemic. Illustrativeroutes of administration include, for example, the nasal, pulmonary,inhalation, intraarterial, intradermal, intralesional, intramuscular,intraperitoneal, intravenous, intrathecal, intravesical, parenteral,rectal, subcutaneous, topical, transdermal, transmucosal, and vaginalroutes.

In some embodiments, the pharmaceutical composition comprising agranuloma fluid, or a composition derived therefrom, is administeredlocally. In some embodiments, the pharmaceutical composition comprisinga granuloma fluid, or a composition derived therefrom, is administeredsystemically.

In some embodiments, the pharmaceutical composition comprising agranuloma fluid, or a composition derived therefrom, is administered bya route of administration selected from the nasal, pulmonary,inhalation, intraarterial, intradermal, intralesional, intramuscular,intraperitoneal, intravenous, intrathecal, intravesical, parenteral,rectal, subcutaneous, topical, transdermal, transmucosal, and vaginalroutes.

In some embodiments, the pharmaceutical composition comprising agranuloma fluid, or a composition derived therefrom, is administered bya route of administration selected from the nasal, pulmonary,inhalation, intraarterial, intradermal, intramuscular, intraperitoneal,intravenous, parenteral, and subcutaneous routes.

In some embodiments, the pharmaceutical composition comprising agranuloma fluid, or a composition derived therefrom, is administered bya route of administration selected from the nasal and pulmonary routes.

In some embodiments, the pharmaceutical composition comprising a cell isadministered locally. In some embodiments, the pharmaceuticalcomposition comprising a cell is administered systemically.

In some embodiments, the pharmaceutical composition comprising a cell isadministered by a route of administration selected from the nasal,pulmonary, inhalation, intraarterial, intradermal, intralesional,intramuscular, intraperitoneal, intravenous, intrathecal, intravesical,parenteral, rectal, subcutaneous, topical, transdermal, transmucosal,and vaginal routes.

In some embodiments, the pharmaceutical composition comprising a cell isadministered by a route of administration selected from the nasal,pulmonary, inhalation, intraarterial, intradermal, intramuscular,intraperitoneal, intravenous, parenteral, and subcutaneous routes.

In some embodiments, the pharmaceutical composition comprising a cell isadministered by a route of administration selected from the nasal,pulmonary, inhalation, intraarterial, intradermal, intramuscular,intraperitoneal, and subcutaneous routes.

In some embodiments, the pharmaceutical composition comprising a cell isadministered by a route of administration selected from the nasal andpulmonary routes.

In some embodiments, the pharmaceutical composition comprising a cell isnot administered intravenously.

5. Further Therapeutic Agents

In some embodiments, the pharmaceutical composition comprises one ormore further therapeutic agents. A further therapeutic agent may be, forexample, a second therapeutic agent, a third therapeutic agent, a fourththerapeutic agent, a fifth therapeutic agent, or an additionaltherapeutic agent beyond a fifth therapeutic agent.

Any suitable further therapeutic agent may be used, and the selection ofthe further therapeutic agent is within the skill of the ordinaryartisan. In some embodiments, the further therapeutic agent is selectedfrom furosemide, aclidinium, albuterol, arformoterol, beclomethasone,budesonide, ciclesonide, clenbuterol, corticosteroids, dexamethasone,fluticasonc, formotcrol, indacatcrol, ipratropium bromide, levalbutcrol,L-argininc, metaproterenol, mometasone, pirbutcrol, salmetcrol,sildenafil, tiotropium, and vilantcrol.

In some embodiments, the further therapeutic agent is serum or plasma.In some embodiments, the further therapeutic agent is platelet-richplasma.

In some embodiments, the further therapeutic agent is a supernatant froma cultured cell, also known as a conditioned medium.

In some embodiments, the further therapeutic agent is a growth factor orcytokine. In some aspects the growth factor or cytokine is selected fromVEGF, adrenomedullin, angiopoietin, autocrine motility factor, bonemorphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF),epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growthfactor (FGF), glial cell line-derived neurotrophic factor (GDNF),granulocyte colony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), growth differentiation factor-9(GDF9), healing factor, hepatocyte growth factor (HGF), hepatoma-derivedgrowth factor (HDGF), insulin-like growth factor (IGF), interleukin-1(IL-1), interleukin-6 (IL-6), keratinocyte growth factor (KGF),migration-stimulating factor, myostatin (GDF-8), nerve growth factor(NGF), neurotrophins, platelet-derived growth factor (PDGF),thrombopoietin (TPO), transforming growth factor alpha(TGF-a),transforming growth factor beta(TGF-(3), tumor necrosis factor-alpha(TNF-a), and placental growth factor (PGF).

If further therapeutic agents are used, the doses of such agents can beobtained from the knowledge of those of skill in the art. For thosefurther therapeutic agents that are approved for clinical use,recommended dosages are described in, for example, Goodman & Gillman'sThe Pharmacological Basis of Therapeutics, Brunton (Ed.) 12th Ed.(2010), McGraw-Hill New York; and Physician's Desk Reference, 67th Ed.(2013), PDR Network, each of which is incorporated by reference in itsentirety.

In some embodiments, a pharmaceutical composition comprising granulomafluid, or compositions derived therefrom, is administered at the sametime as the further therapeutic agent. In some embodiments, apharmaceutical composition comprising cells is administered at the sametime as the further therapeutic agent. When the further therapeuticagent is administered at the same time as these pharmaceuticalcompositions, it can be included in the pharmaceutical composition, oradministered in a separate pharmaceutical composition.

In some embodiments, a pharmaceutical composition comprising granulomafluid, or compositions derived therefrom, is administered at a differenttime from the further therapeutic agent. In some embodiments, apharmaceutical composition comprising cells is administered at adifferent time from the further therapeutic agent.

When the further therapeutic agent is administered at a different timethan the pharmaceutical composition, the further therapeutic agent canbe administered at any suitable time. In some embodiments, the furthertherapeutic agent and the pharmaceutical composition are administeredless than 30 days apart, less than 15 days apart, less than 10 daysapart, less than 5 days apart, less than 1 day apart, less than 12 hoursapart, less than 6 hours apart, less than 3 hours apart, less than 1hour apart, less than 30 minutes apart, or less than 15 minutes apart.

In some embodiments, the further therapeutic agent and thepharmaceutical composition are administered at least 15 minutes apart,at least 30 minutes apart, at least 1 hour apart, at least 3 hoursapart, at least 6 hours apart, at least 12 hours apart, at least 1 dayapart, at least 5 days apart, at least 10 days apart, at least 15 daysapart, or at least 30 days apart.

In some embodiments, the pharmaceutical composition and the furthertherapeutic agent are administered within a time interval that allowsthem to have a synergistic therapeutic effect. This can be useful, forexample, where the pharmaceutical composition or the further therapeuticagent has an undesirable side effect, because the synergy may allow areduction in the dose of either or both.

In some embodiments, pharmaceutical composition is administered to asubject that is also treated with a nasal strip, a mechanicalventilation method, or a hyperbaric oxygen treatment (e.g., in ahyperbaric oxygen chamber).

6. Pharmaceutical Excipients

The pharmaceutical compositions used with the methods provided hereincomprise granuloma fluid, compositions derived from granuloma fluid, orcells. In addition, the pharmaceutical compositions may comprise one ormore pharmaceutical excipients. Any suitable pharmaceutical excipientmay be used, and one of ordinary skill in the art is capable ofselecting suitable pharmaceutical excipients. Accordingly, thepharmaceutical excipients provided below are intended to beillustrative, and not limiting. Additional pharmaceutical excipientsinclude, for example, those described in the Handbook of PharmaceuticalExcipients, Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press;and Controlled Pulmonary Drug Delivery, Smyth and Hickey (Eds.) (2011),Controlled Release Society, each of which is incorporated by referencein its entirety.

In some embodiments, the pharmaceutical composition comprises ananti-foaming agent. Any suitable anti-foaming agent may be used. In someaspects, the anti-foaming agent is selected from an alcohol, an ether,an oil, a wax, a silicone, a surfactant, and combinations thereof. Insome aspects, the anti-foaming agent is selected from a mineral oil, avegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, afatty alcohol wax, a long chain fatty alcohol, a fatty acid soap, afatty acid ester, a silicon glycol, a fluorosiliconc, a polyethyleneglycol-polypropylene glycol copolymer, polydimethylsiloxane-silicondioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethylalcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, andcombinations thereof.

In some embodiments, the pharmaceutical composition comprises acosolvent. Illustrative examples of cosolvents include ethanol,poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, andpropylene glycol.

In some embodiments, the pharmaceutical composition comprises a buffer.Illustrative examples of buffers include acetate, borate, carbonate,lactate, malate, phosphate, citrate, hydroxide, diethanolamine,monoethanolamine, glycine, methionine, guar gum, and monosodiumglutamate.

In some embodiments, the pharmaceutical composition comprises a carrieror filler. Illustrative examples of carriers or fillers include lactose,maltodextrin, mannitol, sorbitol, chitosan, stearic acid, xanthan gum,and guar gum.

In some embodiments, the pharmaceutical composition comprises asurfactant. Illustrative examples of surfactants include d-alphatocophcrol, benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, docusate sodium, glyceryl behenate, glycerylmonooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol,phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitanfatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodiumlauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol)succinate.

In some embodiments, the pharmaceutical composition comprises ananti-caking agent. Illustrative examples of anti-caking agents includecalcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropylcellulose, and magnesium oxide.

In some embodiments, the pharmaceutical composition comprises apropellant. Illustrative examples of propellants include carbon dioxide,chlorodifluoromethane, chlorofluorocarbons, difluoroethane, dimethylether, heptafluoropropane, hydrocarbons (e.g., butane, isobutene,propane), nitrogen, nitrous oxide, and tetrafluoroethane.

Other excipients that may be used with the pharmaceutical compositionsinclude, for example, albumin, antioxidants, antibacterial agents,antifungal agents, bioabsorbablc polymers, chelating agents, controlledrelease agents, diluents, dispersing agents, dissolution enhancers,emulsifying agents, gelling agents, ointment bases, penetrationenhancers, preservatives, solubilizing agents, solvents, stabilizingagents, and sugars. Specific examples o each of these agents aredescribed, for example, in the Handbook of Pharmaceutical Excipients,Rowe et al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press; andControlled Pulmonary Drug Delivery, Smyth and Hickey (Eds.) (2011),Controlled Release Society, each of which is incorporated by referencein its entirety.

In some embodiments, the pharmaceutical composition comprises a solvent.In some aspects, the solvent is saline solution, such as a sterileisotonic saline solution or dextrose solution. In some aspects, thesolvent is water for injection.

In some embodiments, the pharmaceutical composition comprises amacromolecule such as alginic acid, carboxymethylcellulose, carrageenan,chitosan, chondroitin sulfate, collagen, elastin, fibrin, fibronectin,gelatin, heparin sulfate, hyaluronic acid, keratin sulfate, laminin,Matrigeff, methylcellulose, poly(ethylene glycol), poly(lactic acid),poly(lactic-co-glycolic acid), and silk.

In some embodiments, the pharmaceutical compositions are in aparticulate form, such as a microparticle or a nanoparticle.Microparticles and nanoparticles may be formed from any suitablematerial, such as a polymer or a lipid. In some aspects, themicroparticles or nanoparticles are micelles, liposomes, orpolymersomes.

7. Kits

Also provided arc kits for use in the methods of treating EIPH providedherein.

In some embodiments, the kit comprises a pharmaceutical compositioncomprising granuloma fluid, or a composition derived from granulomafluid. Tn some embodiments, the composition is a granuloma powder. Insome embodiments, the powder is lyophilized powder. In some embodiments,the kit further comprises a pharmaceutically acceptable solution forreconstitution of the powder. In some embodiments, the solution isnormal saline. In some embodiments, the kit further comprisesinstructions for the administration of the granuloma fluid orcomposition derived therefrom. In some embodiments, the kit furthercomprises a nasal strip.

In some embodiments, the kit further comprises a device for theadministration of the granuloma fluid or composition derived therefrom.In some embodiments, the device is selected from a nebulizer, a nasalsprayer, a metered dose inhaler, a dry powder inhaler, a cannula, atracheal tube, an endotracheal tube, a tracheostomy tube, an intranasaltube, and a needle. In some embodiments, the device comprises a needlefor injecting the pharmaceutical composition. In some embodiments, thedevice is a device that generates an aerosol using a single-pass, lowshear method. Such low shear methods may be useful for the delivery oflabile molecules. Examples of single-pass, low shear aerosolizationmethods include the SUPRAER™ technology, by KAER Biotherapeutics. Suchmethods are more fully described, for example, in U.S. Pat. Nos.7,802,569; 8,375,987; 8,596,268; 8,616,532; U.S. Pat. Pub. Nos.2007/0144514; 2011/0006129; 2011/0011398; 2011/0011899; 2012/0017899;and 2013/0248615; and Yeates, “SUPRAER™: A New Class of Aerosol DeliverySystem for Biotherapeutics,” On Drug Delivery, 2011, 12-14, each ofwhich is incorporated by reference in its entirety.

In some embodiments, the kit comprises a pharmaceutical compositioncomprising cells. In some embodiments, the cells are stem cells. In someembodiments, the stem cells are mesenchymal stem cells. In someembodiments, the kit further comprises a pharmaceutically acceptablesolution for addition to the cells prior to administration. In someembodiments, the solution is normal saline. In some embodiments, the kitfurther comprises instructions for the administration of the cells.

In some embodiments, the kit further comprises a device for theadministration of the cells. In some embodiments, the device is selectedfrom a nebulizer, a nasal sprayer, a metered dose inhaler, a dry powderinhaler, a cannula, a tracheal tube, an endotracheal tube, atracheostomy tube, an intranasal tube, and a needle. In someembodiments, the device comprises a needle for injecting thepharmaceutical composition. In some embodiments, the device is a deviceadapted to deliver cells by pulmonary or nasal administration. Suitabledevices for delivering cells by pulmonary or nasal administrationinclude nasal sprays (see Shure, Scientific American, 2013, 24:14-15),endoscopes (see Ingenito et al., Cell Transplantation, 2012,21:175-189), intratracheal instillation devices (see Gupta et al., J.Immunol., 2007, 179:1855-1863; and Chang et al., J. Korean Med. Sci.,2014, 29:438-440), transcutaneous transthoracic injection devices (seeLacis et al., J. Sci. Res. Reports, 2014, 3:1780-1792), oraerosolization devices (see Roberts, “Aerosol Delivery of MammalianCells for Tissue Engineering,” Worcester Polytechnic Institute, Masterof Science in Chemical Engineering Thesis, April, 2003; and Kardia etal., J. Aerosol Med. & Pulmonary Drug Deliv., 2013, 26:1-5). Each of thereferences cited in this paragraph is incorporated by reference in itsentirety.

8. Granuloma Fluid

In some embodiments, the pharmaceutical compositions used in the methodsof treatment provided herein comprise granuloma fluid or a compositionderived from granuloma fluid.

8.1. Collection of Granuloma Fluid

The granuloma fluid can be collected by using a hollow device, such as atube, to induce the foreign body reaction, as described in U.S. Pat.Pub. No. 2011/0038903, incorporated by reference in its entirety. Thedevice may have walls that are permeable to the granuloma fluid, therebyallowing the fluid to accumulate inside the hollow space of the device.The granuloma fluid may be harvested by removing the device from theanimal, or by withdrawing fluid from the device while it remains insidethe animal. The device can be implanted at any suitable site within theanimal, including the omentum and subcutaneous tissue.

The device may be formed from any suitable material, including aflexible plastic, a hard plastic, or a metal. In some embodiments, thedevice is formed from a polyethylene or polyvinyl. The ends of thedevice may be sealed, to allow accumulation of fluid inside the device.The walls of the device may have holes, to allow the transport of fluidfrom the cells to the inside of the device.

The size of the device will vary according to the mammal in which it isto be implanted. A person of ordinary skill in the art can readilyselect a device of an appropriate size without undue experimentation,for example, depending on the size of the animal and the amount of fluiddesired. For example, a device for implantation in a rat may have alength of about 10 to about 30 mm, a diameter of about 3.5 to 10.5 mm,and four to twelve holes of about 0.25 to about 0.75 mm. Thesedimensions may be scaled by any appropriate factor to estimate devicesizes for other species. In some embodiments, the scaling may beaccording to the mass or volume of the animal relative to the mass orvolume of another animal. In some embodiments, the scaling may beaccording to the volume of the implantation site relative to the volumeof the corresponding implantation site in another animal.

Granuloma fluid may also be collected by injecting a slurry ofparticles. See U.S. Pat. Pub. No. 2011/0038903, incorporated byreference in its entirety. In some embodiments, the particles arepolydextran particles. In some embodiments, the particles are injectedsubcutaneously. The subcutaneous injection of particles can create a“bladder-like” granulation tissue that can be pierced to withdrawgranuloma fluid.

Granuloma fluid may also be collected by implanting a foam. In someembodiments, the foam is implanted subcutaneously or in the peritonealcavity. The foam serves as a site for the formation of a granuloma. Thegranuloma fluid secreted by the cells is absorbed by the foam and can beretrieved from the foam by further processing, such as by squeezing thefoam.

After collection, granuloma fluid may be processed by any suitablemethod, including centrifugation. In some aspects the granuloma fluid isseparated from cells by centrifugation at 2000 g for 15 minutes, asdescribed in U.S. Pat. Pub. No. 2011/0038903, incorporated by referencein its entirety.

8.2. Characteristics of Granuloma Fluid

The granuloma fluid resembles plasma when analyzed by SDS-PAGE. See U.S.Pat. Pub. No. 2011/0038903, incorporated by reference in its entirety.However, it also contains a variety of potent growth factors that arepresent at low concentrations (relative to albumin and globulins), andtherefore are generally not detectable by SDS-PAGE.

In some embodiments, the granuloma fluid comprises vascular endothelialgrowth factor (VEGF) at a concentration of about 1 ng/mL to about 50ng/mL. In some aspects, the granuloma fluid comprises VEGF at aconcentration of about 2 ng/mL to about 25 ng/mL. In some aspects, thegranuloma fluid comprises VEGF at a concentration of about 2 ng/mL toabout 15 ng/mL. In some aspects, the granuloma fluid comprises VEGF at aconcentration of about 2 ng/mL to about 7 ng/mL. In some aspects, thegranuloma fluid comprises VEGF at a concentration of about 5 ng/mL toabout 10 ng/mL. In some aspects, the granuloma comprises VEGF at aconcentration of about 5 ng/mL. In some aspects, the granuloma comprisesVEGF at a concentration of about 6 ng/mL. In some aspects, the granulomacomprises VEGF at a concentration of about 7 ng/mL. In some aspects, thegranuloma comprises VEGF at a concentration of about 8 ng/mL. In someaspects, the granuloma comprises VEGF at a concentration of about 9ng/mL. In some aspects, the granuloma comprises VEGF at a concentrationof about 10 ng/mL. In some aspects, the granuloma comprises VEGF at aconcentration of about 11 ng/mL. In some aspects, the granulomacomprises VEGF at a concentration of about 12 ng/mL.

In some embodiments, the granuloma fluid comprises VEGF at aconcentration of about 5- to about 100-fold higher than theconcentration of VEGF in the serum or plasma of the animal from whichthe granuloma fluid is derived. In some embodiments, the granuloma fluidcomprises VEGF at a concentration of about 10- to about 80-fold higherthan the concentration of VEGF in the serum or plasma of the animal fromwhich the granuloma fluid is derived. In some embodiments, the granulomafluid comprises VEGF at a concentration of about 20- to about 60-foldhigher than the concentration of VEGF in the serum or plasma of theanimal from which the granuloma fluid is derived. In some embodiments,the granuloma fluid comprises VEGF at a concentration of about 25- toabout 50-fold higher than the concentration of VEGF in the serum orplasma of the animal from which the granuloma fluid is derived.

In some embodiments, the granuloma fluid comprises albumin at aconcentration of about 1 g/L to about 100 g/L. In some embodiments, thegranuloma fluid comprises albumin at a concentration of about 10 g/L toabout 90 g/L. Tn some embodiments, the granuloma fluid comprises albuminat a concentration of about 20 g/L to about 80 g/L. In some embodiments,the granuloma fluid comprises albumin at a concentration of about 30 g/Lto about 70 g/L. In some embodiments, the granuloma fluid comprisesalbumin at a concentration of about 35 g/L to about 50 g/L.

In some embodiments, the granuloma fluid comprises globulins at aconcentration of about 1 g/L to about 100 g/L. In some embodiments, thegranuloma fluid comprises globulins at a concentration of about 10 g/Lto about 90 g/L. In some embodiments, the granuloma fluid comprisesglobulins at a concentration of about 20 g/L to about 80 g/L. In someembodiments, the granuloma fluid comprises globulins at a concentrationof about 30 g/L to about 70 g/L. In some embodiments, the granulomafluid comprises globulins at a concentration of about 26 g/L to about 46g/L.

In some embodiments, the granuloma fluid comprises total protein at aconcentration of about 1 g/L to about 100 g/L. In some embodiments, thegranuloma fluid comprises total protein at a concentration of about 10g/L to about 90 g/L. In some embodiments, the granuloma fluid comprisestotal protein at a concentration of about 20 g/L to about 80 g/L. Insome embodiments, the granuloma fluid comprises total protein at aconcentration of about 40 g/L to about 80 g/L. In some embodiments, thegranuloma fluid comprises total protein at a concentration of about 60g/L to about 80 g/L.

In some embodiments, the granuloma fluid is processed by a method thatalters the amount of one or more of its components. Any suitableprocessing method may be used. Illustrative processing methods includedialysis, size filtration (such as centrifugal filtration),chromatography (such as size exclusion chromatography), andelectrophoresis.

In some embodiments, the granuloma fluid is processed to alter theamount of albumin. The molecular weight of albumin varies by species,but generally ranges from about 55 kDa to about 80 kDa, or about 65 kDato about 70 kDa. Accordingly, the amount of albumin in the granulomafluid can be reduced by performing one or more fractionation steps thatremove proteins in the molecular weight range appropriate for thespecies from which the granuloma fluid is collected. In some aspects,the amount of albumin in the processed granuloma fluid is reduced by atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or at least about 95% in comparisonto the granuloma fluid as harvested from the animal.

In some embodiments, the granuloma fluid is processed to alter theamount of VEGF. The amount of VEGF in the granuloma fluid can be reducedby, for example, molecular weight fractionation or by affinity-basedmethods using reagents that are specific for VEGF. In some aspects, theamount of VEGF in the processed granuloma fluid is reduced by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 95% in comparison tothe granuloma fluid as harvested from the animal.

Reducing the amount(s) of albumin in the granuloma fluid will alsoreduce the amount of total protein in the fluid. Reduction of the amountof total protein may be useful, for example, to reduce the amount ofanti-foaming agent that is used in certain formulations.

Cells for the production of granuloma fluid can be obtained from anysuitable mammal. Suitable mammals include, for example, horses, dogs,cats, camels, pigs, cows, goats, llamas, sheep, mice, rats, rabbits, andhumans. In some aspects, the mammal is a horse. In some aspects themammal is a human.

9. Cells

In some embodiments, the pharmaceutical compositions used in the methodsof treatment provided herein comprise cells. In some embodiments, thecells are stem cells. In some embodiments the stem cells are embryonicstem cells. In some embodiments the stem cells are adult stem cells. Insome embodiments, the stem cells are mesenchymal stem cells.

In some embodiments, the cell is selected from a macrophage, alymphocyte, a neutrophil, an eosinophil, a multinucleated giant cell,and a fibroblast, and combinations thereof.

In some embodiments, the cells are cells collected with the granulomafluid. Any cell proximal to the site of the granuloma may be useful withthe methods of treatment provided herein.

In some embodiments, the foreign body used to collect the granulomafluid may also be used to collect cells. The cells may be isolated fromthe proximity of the foreign body, or from the portion of the foreignbody used to collect the granuloma fluid (e.g., the cavity of a tube).Collection of cells from a granuloma is discussed, for example, in U.S.Pat. Pub. No. 2011/0038903, incorporated by reference in its entirety.

Mesenchymal stem cells can be identified by the presence of a variety ofmarkers, and by canonical pathways of differentiation. In someembodiments, the mesenchymal stem cells express CD105, CD73, and CD90,and do not express CD45, CD34, CD14, CD11b, CD79a, CD19, or HLA-DR. SeeDominici et al., Cytotherapy, 2006, 8:315-317, incorporated by referencein its entirety.

In some embodiments, the mesenchymal stem cells do not express CD31 orCD133. See Lodie et al., Tissue Engineering, 2002, 8:739-751. In someembodiments, expression of CD44 depends on the serum concentration. Seeid.

In some embodiments, the mesenchymal stem cells express SH2, SH3, CD29,CD44, CD71, CD90, CD106, CD120a, CD124, and are negative for markers ofthe hematopoietic lineage, such as CD14, CD34, and CD45. See Pittengeret al., Science, 1999, 284:143-147.

In some embodiments, the mesenchymal stem cells are capable ofdifferentiating into osteoblasts, adipocytes, and chondrocytes whenexposed to the appropriate medium.

In some embodiments, the mesenchymal stem cells are derived fromadipose, bone marrow, dental pulp, colon, heart, liver, lung, tonsil,umbilical cord, amnion, or fetal liver. Markers for the identificationof these cell types arc known in the art and provided, for example, inTable 1 of Signore et al., J. Cell. Physiol., 2011, 227:3291-3300,incorporated by reference in its entirety.

In some embodiments, the mesenchymal stem cells support hematopoieticactivity and have immunomodulatory activity. See Signore et al., J Cell.Physiol., 2011, 227:32913300, incorporated by reference in its entirety.

In some embodiments, the mesenchymal stem cells are lung mesenchymalstem cells as described, for example, in Ingenito et al., CellTransplantation, 2012, 21:175-189, incorporated by reference in itsentirety.

10. Examples Example 1: Treatment of Horse with Granuloma Fluid

The present example provides methods and results for treating a horsesuffering from exercise-induced pulmonary hemorrhage with a compositionof provided herein.

HORSE #1, a four-year old thoroughbred, was evaluated prior to treatmentfollowing an easy gallop. Evaluation was by endoscopic examination ofthe trachea according to Hinchcliff, et al., 2005, 1 Am. Vet Med. Assoc.227:768-74.

Grade 0: No blood detected in the pharynx, larynx, trachea, or mainstembronchi.

Grade 1: Presence of one or more flecks of blood or two or fewer short(less than one-quarter the length of the trachea) and narrow (less than10% of the tracheal surface area) streams of blood in the trachea ormainstem bronchi (the two airways the trachea splits into; these carryair to and from the right and left lungs) visible from the trachealbifurcation.

Grade 2: One long stream of blood (more than one-half the length of thetrachea) or more than two short streams of blood occupying less thanone-third of the tracheal circumference.

Grade 3: Multiple, distinct streams of blood, covering more thanone-third of the tracheal circumference, with no blood pooling at thethoracic inlet.

Grade 4: Multiple, coalescing streams of blood covering more than 90% ofthe tracheal surface with blood pooling at the thoracic inlet.

Prior to treatment, HORSE #1 showed a bleeding score of 3 out of 4 afteran easy gallop. The score was assigned by a practitioner using the scaleabove.

HORSE #1 received a first treatment including a full dose (10 cc) ofgranuloma powder (VivaStem-Equine Injection Kit; VivaStem LaboratoriesLLC) dissolved in saline solution and administered by nebulizer(Flexineb, Nortev) to the lungs. Following treatment at 24 hours, HORSE#1 jogged for one mile. At 48 hours, HORSE #1 galloped for 1.5 miles. At72 hours, HORSE #1 galloped for 1.5 miles. No bleeding was observed 24,48, and 72 hours following treatment. A grade of 0/4 was assigned.

Eight days later, HORSE #1 received a second treatment of 1Occ dissolvedgranuloma powder, as described above. Following treatment at 24 hours,HORSE #1 had a mild jog. At 48 hours, HORSE #1 galloped for 1.5 miles.At 72 hours, HORSE #1 galloped for 1.5 miles. On endoscopic evaluationfollowing treatment, no bleeding was observed 24, 48, and 72 hoursfollowing treatment. A grade of 0/4 was assigned.

Two weeks later, HORSE #1 was evaluated a third time, following a twominute effort at an average of about 15 seconds per ⅛ mile. Onendoscopic evaluation, no bleeding was observed, and an endoscopic scoreof 0 was assigned.

After another week, HORSE #1 was evaluated a fourth time, following atwo minute effort at an average of about 15 seconds per ⅛ mile. Onendoscopic evaluation, no bleeding was observed, and an endoscopic scoreof 0 was assigned.

This example demonstrates that one and two administrations of acomposition provided herein reduced bleeding in an exercise-inducedpulmonary hemorrhage subject when evaluated by pulmonary endoscopy. Theendoscopic score was reduced from ¾ on an initial evaluation to 0/4 onthe standard scale used in the field.

11. Equivalents

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in this application, in applications claiming priority fromthis application, or in related applications. Such claims, whetherdirected to a different invention or to the same invention, and whetherbroader, narrower, equal, or different in scope in comparison to theoriginal claims, also are regarded as included within the subject matterof the inventions of the present disclosure.

What is claimed:
 1. A method of treating exercise-induced pulmonaryhemorrhage in a mammal in need thereof, comprising administering aneffective amount of a pharmaceutical composition comprising granulomafluid, or a composition derived therefrom, to the mammal, wherein thegranuloma fluid or composition derived therefrom comprises stem cells inan amount of about 5×10⁴ to about 5×10¹¹ cells.
 2. The method of claim1, wherein the pharmaceutical composition is administered by a route ofadministration selected from the nasal and pulmonary routes.
 3. Themethod of claim 1, wherein the subject is selected for treatment basedon epistaxis, endoscopy, broncho-alveolar lavage, biopsy, radiograph,pulmonary scintigraphy, a history of exercise-induced pulmonaryhemorrhage, or an elevated pulmonary blood pressure.
 4. The method ofclaim 1, wherein the mammal is a horse, a dog, a camel, a monkey, a cat,a pig, a cow, a goat, a llama, a sheep, a mouse, a rat, a rabbit, or ahuman.
 5. The method of claim 1, wherein the granuloma fluid, orcomposition derived therefrom, is administered to the subject at a dosethat is equivalent to about 1 mg to about 20 g of granuloma powder. 6.The method of claim 1, wherein the granuloma fluid, or compositionderived therefrom, is administered to the subject at a dose that isequivalent to about 200 mg to about 5 g of granuloma powder.
 7. Themethod of claim 1, wherein pharmaceutical composition is administeredwith an additional therapeutic agent.
 8. The method of claim 7, whereinthe additional therapeutic agent is administered within 7 days of thepharmaceutical composition.
 9. The method of claim 7, wherein thepharmaceutical composition comprises the additional therapeutic agent.10. The method of claim 9, wherein the additional therapeutic agent isfurosemide, aclidinium, albuterol, arformoterol, beclomethasone,budesonide, ciclesonide, clenbuterol, corticosteroids, dexamethasone,fluticasone, formoterol, indacaterol, ipratropium bromide, levalbuterol,L-arginine, metaproterenol, mometasone, pirbuterol, salmeterol,sildenafil, tiotropium, and vilanterol, a cell, platelet-rich plasma,conditioned medium, or nasal strips.
 11. The method of claim 1, whereinthe composition derived from granuloma fluid is selected from agranuloma powder, a concentrated granuloma fluid, and a dilutedgranuloma fluid.
 12. The method of claim 1, wherein the pharmaceuticalcomposition is administered by a device selected from a nebulizer, anasal sprayer, a metered dose inhaler, a dry powder inhaler, a cannula,a tracheal tube, an endotracheal tube, a tracheostomy tube, anintranasal tube, an endoscope, a transcutaneous transthoracic injectiondeice, a needle, or a single-pass low aerosolization method.
 13. Themethod of claim 1, wherein the granuloma fluid, or composition derivedtherefrom, is from the mammal.
 14. The method of claim 1, wherein thegranuloma fluid, or composition derived therefrom, is from a mammal thatis of the same species as the subject.
 15. The method of claim 1,wherein the granuloma fluid, or composition derived therefrom, is from amammal that is of a different species from the subject.